An important player in the calcium induced calcium release process is the "cardiac" ryanodine receptor, RyR2. This is a large protein (500 kD) which forms a tetrameric channel 30x30x15 nm in size. The trans-membrane channel is believed to be formed by the C-terminal. Using standard approaches, we have created and examined ES cells lacking RyR2 to show that this protein is critical in the regulation of heart rate during development. RyR2 null mice produce an embryonic-lethal phenotype by 10 dpc. Functional studies are on-going to determine the reason for the lethality, as it does not appear to be linked to a 'classic' EC coupling mechanism. We have also established a tripartite mouse model system to study the in-vivo function of this channel using a transgenic approach to produce an inducible, tissue-restricted null background by in adult mice hearts. To avoid embryonic lethality, we prepared several transgenic lines that would permit conditional and inducible gene targeting, limited to specific cardiac lineages (e.g. ventricular myocytes) and inducible at a desired developmental stage (particularly in the adult). The tools to accomplish this were the Cre recombinase - LoxP recombination system and the tetracycline trans-activator system. The mutant mice containing the RyR2 targeting vector are now being crossed with the regulatory transgenic mice to permit regulated knock-out of the transgene in the adult mouse. Their phenotype, both of individual cardiac myocytes and at a whole animal level, will be examined to determine the essential role of the RyR2 protein on EC coupling. Ultimately, rescue of the null-phenotype will be achieved through targeting of the floxed sequences with chimeric cDNA constructs to permit the endogenous transcriptional machinery of the RyR2 to transcribe mutant forms of the protein. Proof of this system has already been generated in ES cells null for the RyR2 gene, and we will exploit this system to generate transgenic mice with mutant forms of the ryanodine receptor to study specific questions related to the structure and function of the RyR2 protein.